Abstract ?-Secretase represents a novel class of proteases that cleaves substrates within the transmembrane domain. Moreover, ?-secretase has a major player in Alzheimer?s disease (AD). Therefore, ?-secretase has emerged to the forefront of basic and translational research. ?-Secretase interacts with multiple regions of APP (amyloid precursor protein) substrate. The precise mechanism how these interactions are regulated remains to be investigated. We have discovered a substrate inhibitory domain (ASID) within APP that negatively modulates ?-secretase activity. Furthermore, altering the function of ASID can lead to a potential pathological outcome, such as is the case of the familiar APP Flemish mutation. The objectives of this application are to determine how a substrate inhibitory domain (ASID) within APP interacts with ?-secretase, to examine the function of ASID in Notch1 cleavage and to determine the role of ?-secretase cleavage in the regulation of ?-secretase. We will map the APP ASID binding sites within the ?-secretase complex determine the underlying mechanism of ASID in the regulation of ?-secretase. We will investigate the role of the Notch substrate inhibitor domain and elucidate its regulatory mechanism. Lastly, we will determine the function of ADAM10 cleavage in the regulation of ?-secretase activity for A? production and examine the role of ADAM10 mutations associated with late onset AD. Overall, this proposal uses innovative approaches to address an unprecedented mechanism in the regulation of ?-secretase, which represents a topic highly significant in AD pathogenesis and therapeutic development.